Analyte extraction apparatus and method

ABSTRACT

Disclosed is a method and apparatus for determining a concentration of a glycopeptide antibiotic containing a phenol moiety such as Vancomycin in a complex sample matrix by extracting the glycopeptide antibiotic from a metered portion of the complex sample matrix by exposing said metered portion to an extraction material having an affinity with the glycopeptide antibiotic; and exposing the extraction material to a metered portion of an eluent for releasing the glycopeptide antibiotic from the extraction material; and by determining a concentration of the glycopeptide antibiotic by adding a Gibbs reagent (2,6 dichloroquinone-4chloroimide) to the metered portion of the complex sample matrix or the eluent; activating the Gibbs reagent and, after the reaction between the activated Gibbs reagent and the antibiotic has stabilized; detecting the reaction product of the activated Gibbs reagent and the antibiotic in said eluent; and determining the concentration of the antibiotic in the complex sample matrix from the detected reaction product. A method of designing a personalized drug administration regime using the thus obtained concentration is also disclosed.

FIELD OF THE INVENTION

The present invention relates to a method and apparatus for improvingthe automation of cell lysis for use in the extraction and measurementof analyte concentrations in biological samples. In particular, thisinvention provides a means for automatically diluting and lysing bloodcells prior to Vancomycin extraction and analysis.

BACKGROUND OF THE INVENTION

Since their discovery, antibiotics have become critical in the fightagainst infectious diseases caused by bacteria. However their extensiveand inappropriate use is one of the biggest drivers of drug resistance;see. e.g., Davies, S., Infections and the Rise of AntimicrobialResistance, Annual Report of the Chief Medical Officer—Volume Two, 2011;Chan, M., Combating Antimicrobial Resistance: Time for Actionconference, WHO, 2013; and J. W. Ndieyira et al. in NatureNanotechnology 3(11), pages 691-696, 2008.

The emergence of new infections and the re-emergence of old enemies,such as antibiotic-resistant hospital ‘superbugs’, methicillin-resistantStaphylococcus aureus (MRSA) and Vancomycin-resistant Enterococci (VRE),and the associated increase in fatalities, is a major global healthcareproblem, costing the NHS alone £1bn per annum. At the same time, theantibiotic-pipeline has nearly dried up with just a few new classes ofantibiotics discovered over the last forty years; see e.g. M. A Cooperet al., Nature 472, page 32, 2011 and M. S. Butler et al. in Journal ofAntibiotics 64(6), pages 413-425, 2011. This lack of new potent drugsputs us at risk of returning to the pre-antibiotic era of untreatableinfections, or as the WHO recently forecasted “the world is heading fora post-antibiotic era”. Therefore, there is an urgent need for tools toimprove the stewardship of antibiotics to minimise the evolutionarypressure on bacteria, which inevitable leads to antimicrobial drugresistance.

In clinics, the administrated drug concentrations for many drugs,including (glycopeptide) antibiotics, relies on a combination ofclinical judgment, averaged pharmacokinetic models and subjectivemethods of assessing the effect of the therapy, resulting in an inherentvariability in the quality of therapy. The administration of manytherapeutic drugs, for example a glycopeptide antibiotic such asVancomycin, is routinely guided by therapeutic drug monitoring (TDM)which can be significantly improved with more frequent monitoring,particularly in the first 24 hours of administration. Conclusively,there is an urgent need for new technologies to tailor treatments toindividual patients' needs, particularly patient populations at risk,such as paediatrics, obese, elderly, immune-compromised, intensive careunit (ICU) and oncology patients.

Furthermore, in the special case of the glycopeptide antibioticVancomycin with its possible nephrotoxicity, patients which receivingconcomitant nephrotoxic drugs are at high risks of undesirable toxicside effects. Therefore especially for those patients, there is anurgent need for monitoring drug levels at the point-of-care withinminutes in order to personalise and optimise therapy at the patientlevel. This need has been recognized as early as 2002; see C. M. Tobinet al., Journal of Antimicrobial Chemotherapy, 50(5), pages 713-718,2002. The British National Formulary (BNF) recommends peak serum valuesfor Vancomycin to be in the range of 25 to 40 μg/ml which corresponds to17.3 to 27.6 μM of Vancomycin, and trough values should be in the rangeof 10 to 15 μg/ml and 15 to 20 for complicated infections whichcorresponds to 6.9 to 10.4 μM and 10.4 to 13.8 μM Vancomycinrespectively. For paediatrics the peak serum values can reach 60 μg/ml,which corresponds to 41.4 μM of Vancomycin, and trough values aretypically measured in the range of 5 to 10 μg/ml which corresponds to3.5 to 6.9 μM Vancomycin.

The state-of-the-art in clinics for therapeutic Vancomycin monitoringrequires extensive sample preparation and often the samples have to besent to a specialised laboratory with trained staff. This is expensive,laborious, time-consuming, and leads to inevitable delays between testsand results, which means important therapeutic decisions are delayed andpatient pathways can be slow and cumbersome, as also recognized by M. ACooper et al., Nature 472, page 32, 2011 and by C. M. Tobin et al.,Journal of Antimicrobial Chemotherapy, 50(5), pages 713-718, 2002.

Furthermore, routine drug monitoring only measures the total antibioticconcentration even though protein binding varies dramatically (10-82%protein bound) with 55% often quoted as the mean fraction bound; seee.g. Zeitlinger et al., Antimicrobial Agents and Chemotherapy 55(7),pages 3067-3074, 2011. Since measurement of the free Vancomycinconcentration requires several preparation steps and is consequentlyvery time consuming and expensive, it is not routinely performed inhealth care facilities see e.g. Berthoin et al., International Journalof Antimicrobial Agents 34(6), pages 555-560, 2009. This is problematicas it is generally accepted that only the free drug fraction ispharmacologically active and the fraction of drug bound to serumproteins is inactive. Moreover, studies have suggested that thecorrelation between free and total fraction is poor, see e.g. Estes &Derendorf, European Journal of Medical Research, 15(12), pages 533-543,2010 and Butterfield et al. Antimicrobial agents and Chemotherapy 55(9),pages 4277-4282 2011.

The current gold standards in therapeutic Vancomycin drug monitoringare:

-   -   fluorescence polarisation immunoassay (FPIA), such as the        “FLx/TDx” from Abbott Diagnostics, UK.    -   homogenous enzyme immunoassay, such as the “ONLINE TDM        Vancomycin assay” from COBAS®, Roche, CH.

In 2002, NHS Bristol launched a survey to study Vancomycin therapeuticdrug monitoring (TDM) as disclosed by C. M. Tobin et al., Journal ofAntimicrobial Chemotherapy, 50(5), pages 713-718, 2002. They questioned310 participants from UK NHS hospitals, UK public health laboratories,UK private hospitals and other European and non-European hospitals.According to this survey, the cost of a Vancomycin assay includingtaking blood, transport to the laboratory (since microbiologydepartments are still the main providers of assays), time for paperwork,running the assay, result reporting and interpretation was estimated tocost around £35, which exceeds the drug costs for twice-daily 1 gintravenous dosing.

Moreover the Tobin study showed that around 65% of all assays onlyreceived their results in one day. Almost exclusively, 97% of therespondent were using the fluorescence polarisation immunoassay (FPIA)purchased from Abbott Diagnostics, Maidenhead, UK.

Furthermore a recent study published by Touw et al. in European Journalof Hospital Pharmacy Science, page 13, 2007, presented the results ofcost-effectiveness study of therapeutic drug monitoring (TDM). Theirstudy published results on aminoglycoside and Vancomycin treatments andshowed a statistically significant higher death rate (6.3%), longerstays in hospitals (12.3%), higher hearing loss (46.3%) and renalimpairment (34.0%), and consequently higher total charges (6.3%) inhospitals that did not have pharmacist-managed therapies, which includesTDM combined with results interpretation by using mathematical andpharmacokinetic models and then advising the physicians correspondingly.Conclusively they recommend that Vancomycin therapy is guided by TDM,especially in patient populations at risk such as ICU patients, oncologypatients and patient receiving concomitant nephrotoxic medicines.

It is therefore clear that there is a long-felt need for more facile andaccurate detection of antibiotic levels, and in particular glycopeptideantibiotic levels in the complex sample matrix of a patient at the pointof care, and in particular Vancomycin levels, to facilitate rapid TDM atthe point-of-care. In particular, a need exists to accurately detectfree (unbound) and bound fractions of glycopeptide antibiotics in suchcomplex sample matrices at the patient's point of care.

SUMMARY OF THE INVENTION

The present invention seeks to provide a method and apparatus forautomating the detection of glycopeptide antibiotics comprising a(poly)phenolic functional group, e.g. Vancomycin, that can producereliable detection of levels of such glycopeptide antibiotics in acomplex sample matrix of a patient, e.g. blood, plasma or serum, at thepoint-of-care.

In accordance with an aspect of the present invention, there is provideda method of determining a concentration of an antibiotic containing aphenol moiety, i.e. at least one phenol moiety, in a complex samplematrix. The method comprises extracting the glycopeptide antibiotic froma metered portion of the complex sample matrix by exposing said meteredportion to an extraction material having an affinity with theglycopeptide antibiotic; and exposing the extraction material to ametered portion of a first eluent being a first organic solvent or asecond eluent being a second organic solvent/water mixture for releasingthe glycopeptide antibiotic from the extraction material; anddetermining a concentration of the glycopeptide antibiotic by adding anexcess amount of Gibbs reagent (2,6 dichloroquinone-4-chloroimide)relative to the amount of the glycopeptide antibiotic to the meteredportion of the first or second eluent; activating the Gibbs reagentunder alkaline conditions and, after the reaction between the activatedGibbs reagent and the antibiotic has stabilized; detecting the reactionproduct of the activated Gibbs reagent and the antibiotic in said firstor second eluent; and determining the concentration of the antibiotic inthe complex sample matrix from the detected reaction product, saidconcentration being the concentration of unbound antibiotic in thecomplex sample matrix in case of the first eluent; and the concentrationof bound antibiotic in the complex sample matrix in case of the secondeluent.

It has surprisingly been found that a glycopeptide antibiotic containingphenol moieties such as Vancomycin can be effectively extracted from acomplex sample matrix using an extraction assay that has been previouslyused for the extraction of Propofol from such a sample matrix; see WO2012/049486 A1.

Moreover, it has surprisingly been found that an appropriate choice ofeluent can selectively extract the free or bound glycopeptide antibioticfraction using such an extraction assay, as it has been found that theaffinity of the glycopeptide antibiotic with the extraction material inthe assay depends on whether the glycopeptide antibiotic is bound to theextraction material only as is the case for the free fraction, orwhether the glycopeptide antibiotic is bound to the extraction materialand/or to matter extracted from the complex sample matrix such ascellular material, extracellular material, e.g. proteins, fatty acids,glucose, urea, lactic acid, electrolytes and so on, as is the case forthe bound fraction. Therefore, the free and bound fraction ofglycopeptide antibiotics such as Vancomycin can be accurately determinedwith a single assay.

In addition, it has surprisingly been found that the Gibbs reaction canbe used for a phenolic glycopeptide antibiotic such as Vancomycin inorder to obtain a reaction product that can be electrochemically orcolorimetrically detected. This is unexpected since thepara-unsubstituted position (position 6) of the dihydroxy benzene (whichis the 7^(th) residue of the glycopeptide antibiotics) in suchantibiotics, and in the particular case of Vancomycin, is stericallyhindered, which prima facie suggests a low chance of successfullycompleting the Gibbs reaction for such antibiotics. This method enablesa much better control of drug delivery at the point-of-care and leads tothe improved management of infections, patient treatment, safety andhealth outcomes, as well as curbs the incorrect usage of antibioticsthat fuels resistance. It provides frequent, fast and accurateinformation throughout patient treatment as well as reduces thetherapeutic decision time.

At this point, it is noted that the abstract of Rao et al. in Indianpharmacist 2(9), page 59-61, 2003 and the thesis titled “Studies onDevelopment and Validation of Analytical Procedures for the Estimationof Selected Drugs through new Rp-HPLC methods” by K. Bala MuraliKrishna, Acharya Nagarjuna University, chapter 3 on page 69, 2010[http://shodhganga.inflibnet.ac.in/handle/10603/8209] suggest theformation of the Vancomycin-Gibbs reaction product by performing theGibbs reaction in an acidic medium. They obtained a yellow colouredproduct with maximal absorbance wavelength (λ_(max)) at 400 nm and 460nm respectively in the aforementioned citations. However, the presentinventors have found that such a yellow coloured product corresponds tothe activated Gibbs reagent rather than the Gibbs-Vancomycin reactionproduct.

In contrast, the present inventors have realized that the desiredGibbs-Vancomycin reaction product may be formed under alkaline reactionconditions, which produces a distinct purple/magenta product that has amaximal absorbance wavelength (λ_(max)) in the range of 580-600 nm.

Embodiments of the present invention enable detection of glycopeptideantibiotics containing phenol moieties such as Vancomycin at clinicallyrelevant concentrations (3.5-27.5 μM or 5-40 μg/ml) with lower detectionlimits comparable to the aforementioned gold standard Roche Cobas Vanc2assay (1.2 μM), with the clear benefit that drug quantification isachieved within minutes, compared to several hours when having to sendsamples for analysis with laboratory-based assays.

The first and/or the second organic solvent may be methanol.

In an embodiment, said exposing step is performed with the first eluent,the method further comprising repeating the exposing and determinationsteps with the second eluent. In this manner, both the free and boundfractions of the glycopeptide antibiotic in the complex sample matrixcan be determined from the first and second eluents respectively.

In a particularly advantageous embodiment, the method further comprisesexposing the extraction material to at least one washing step prior toexposing the extraction material to said metered portion of the firstand/or second eluent to remove weakly bound impurities from theextraction material in order to remove weakly bound impurities from theextraction material that can interfere with the Gibbs reaction.

The complex sample matrix may comprise matter such as cellular andextracellular material as previously explained, in which case theexposing step may comprise providing a known quantity of the complexsample matrix; diluting said known quantity with a lysing agent, saidlysing agent having an osmotic pressure lower than the osmotic pressureof the cellular material; and subsequently feeding the diluted knownquantity through a filtering stage comprising the extraction materialafter a predetermined amount of time to separate the glycopeptideantibiotic from the destabilized matter, e.g. cellular and extracellularmaterial, by adhering the glycopeptide antibiotic to the extractionmaterial.

The extraction material may further comprise a sorbent material formechanically lysing the cellular material, said sorbent material havingan affinity for binding the glycopeptide antibiotic such as a reversephase sorbent material, such as C8, C12, C18 or Strata-X.

In an embodiment, the detecting step comprises electrochemicallydetecting the reaction product of the activated Gibbs reagent and theglycopeptide antibiotic.

In an alternative embodiment, the detecting step comprisescolorimetrically detecting the reaction product of the activated Gibbsreagent and the glycopeptide antibiotic. This is particularlyadvantageous as the reaction product can be readily detected without theneed for any further reaction. For instance, for Vancomycin, saidcolorimetrically detecting comprises measuring the intensity of theabsorption at 589 nm.

The Gibbs reaction may be performed at any suitable point in time, e.g.following said extracting step.

The complex sample matrix is not necessarily retrieved from a (human)patient. In an alternative embodiment, the complex sample matrix isobtained from a food product such as milk or meat, which facilitates therapid determination of glycopeptide antibiotic levels in such foodproducts, which improves the ease with which food standards may becontrolled, as the presence of (excessive amounts of) glycopeptideantibiotics in such food products is prohibited in most jurisdictions.

In accordance with another aspect of the present invention, there isprovided a method of providing a personalized administration regime of aglycopeptide antibiotic containing a phenol moiety for a patient, themethod comprising determining the concentration of the glycopeptideantibiotic in a complex sample matrix of said patient in accordance withan embodiment of the glycopeptide antibiotic concentration determiningmethod of the present invention; deriving metabolic characteristics ofthe glycopeptide antibiotic for said patient from the determinedconcentration of the glycopeptide antibiotic in said complex samplematrix; and providing said personalized glycopeptide antibioticadministration regime based on said metabolic characteristics.

This enables the drug dose to be administered, e.g. titrated, to thedesired target concentration according to patient's individual drugadsorption, distribution, metabolism and excretion, detect accumulationor changes in the drug clearance rate and provide early detection offaults in the drug delivery system. Consequently monitoring drug levelsat the point-of-care will be a key tool in individualising, optimisingand managing therapy at the patient level, and therefore provide animportant step from passive “monitoring” towards (pro-)active managementof drug concentrations in patients.

In an embodiment, the determining step comprises periodicallydetermining the concentration of the glycopeptide antibiotic in acomplex sample matrix, wherein said deriving step comprises derivingsaid metabolic characteristics from a trend in the periodicallydetermined concentrations.

In accordance with yet another embodiment of the present invention,there is provided an apparatus for automatically extracting of aglycopeptide antibiotic containing a phenol moiety from a complex samplematrix comprising matter such as cellular and extracellular material,the apparatus comprising a sample reception stage having on output forproviding a defined quantity of the complex sample matrix; a mixingstage having a first input in fluidic connection with the output of thesample reception stage, a second input for receiving a lysing agent andan output for providing a mixture of the defined quantity of the complexsample matrix and the lysing agent;

a delay stage having an input in fluidic connection with the output ofthe mixing stage and an output for providing the delayed mixture of thedefined quantity of the complex sample matrix and the lysing agent; afiltering stage comprising a sorbent material for mechanically lysingthe cellular material, said sorbent material having an affinity forbinding of the glycopeptide antibiotic, said filtering stage having aninput in fluidic connection with the output of the delay stage; and acontroller for controlling the flow rate of the mixture of the definedquantity of the complex sample matrix and the lysing agent through saiddelay stage, wherein the apparatus further comprises a colorimetricmeasurement stage in fluid connection with the filtering stage andarranged to determine the colorimetric spectrum of an eluent receivedfrom the filtering stage including a reaction product of theglycopeptide antibiotic and the activated Gibbs reagent; and a processorcoupled to the colorimetric measurement stage adapted to derive aconcentration of the glycopeptide antibiotic in the complex samplematrix from a specific wavelength in the colorimetric spectrum.

Such an apparatus can automatically detect the level of a glycopeptideantibiotic with one or more phenolic moieties such as Vancomycin in sucha complex sample matrix, thus facilitating an improved control of drugdelivery at the point-of-care and improved management of infections,patient treatment, safety and health outcomes, as well as the curbing ofthe incorrect usage of antibiotics that fuels resistance by providingfrequent, fast and accurate information throughout patient treatment andreduce the therapeutic decision time. The apparatus provides combinedosmotic and mechanical lysis inside the apparatus and, preferentiallyachieving the mechanical lysis by passing the diluted sample across asorbent with affinity for the analyte of interest. This approachenhances the efficiency of the release of a glycopeptide antibioticcontaining phenol moieties such as Vancomycin onto the sorbent. Inaddition, the apparatus is constructed such that any sample materialbeing accumulated on top of the sorbent is still available for thesubsequent glycopeptide antibiotic extraction and detection/measurement.

In addition, the novel apparatus provides for more efficient use as itallows the injection of a whole complex sample matrix into theinstrument without the requirement for pre-treatment or dilution. Thishas the important advantage that the actual volume of lysing agent useddoes not need determining, as the metering is performed on the actualcomplex sample matrix, such that the amount of glycopeptide antibioticextracted from the sample diluted by the lysing agent directlycorresponds to the amount of glycopeptide antibiotic present in thecomplex sample matrix; i.e. the amount of lysing agent used isirrelevant other than for the purpose of ensuring that the cellularmaterial is sufficiently destabilized, e.g. ruptured.

In an embodiment, the specific wavelength is in the range of 580-600 nm,and preferably is 589 nm in case of the glycopeptide antibioticcontaining phenol moieties being Vancomycin.

The sample reception stage may comprise a metering device such as arotary valve for extracting a known quantity of a complex sample matrixreceived at the input of the sample reception stage. This reduces therequired amount of reagents such as the lysis agent as the sampleevaluation is performed on a portion of the sample only.

The apparatus may further comprise a fluid reservoir in fluid connectionwith the filtering stage for providing the filtering stage with awashing agent for removing materials that would interfere with thesubsequent extraction of the glycopeptide antibiotic from the sorbentmaterial. This further improves the accuracy of the glycopeptideantibiotic, e.g. Vancomycin, detection with the apparatus.

The filter stage may be comprised in a solid phase extraction cartridgeto facilitate reusability of the apparatus of the present invention atminimal cost overhead as such reusable cartridges can be manufactured ata small cost.

In an embodiment, the sorbent material is a reverse phase sorbentmaterial, such as C8, C12, C18 or Strata-X as such materials have anaffinity for binding glycopeptide antibiotics containing one or morephenolic moieties. Strata-X is particularly suitable for bindingVancomycin.

BRIEF DESCRIPTION OF THE EMBODIMENTS

Embodiments of the invention are described in more detail and by way ofnon-limiting examples with reference to the accompanying drawings,wherein:

FIG. 1 schematically depicts a flowchart of an embodiment of themeasurement method of the present invention incorporating the novelsample preparation approach;

FIG. 2 schematically depicts an embodiment of the device of the presentinvention incorporating the novel sample preparation method;

FIG. 3 depicts absorption characteristics of the reaction of Vancomycinwith an equimolar amount of Gibbs reagent;

FIG. 4 (A) shows the theoretical prediction of the isotopic pattern ofmass from the novel coupling product (C₇₂H₇₆Cl₄N₁₀O₂₅) based on 1:1stoichiometric ratio of Vancomycin:Gibbs under alkaline condition,whereas (B) shows the experimentally measured mass spectrum of the novelreaction product;

FIG. 5 depicts ¹H-NMR spectra of the Vancomycin-Gibbs reagent reactionproduct for a number of Vancomycin:Gibbs molar reagent ratios;

FIG. 6-7 depict absorption characteristics of the reaction of Vancomycinextracted from a complex sample matrix with the activated Gibbs reagent;

FIG. 8 depicts a fit of measured differential absorbance as determinedusing an embodiment of the colourimetric method of the present inventionagainst known vancomycin concentrations in eluent solutions;

FIG. 9 depicts absorption characteristics of the reaction of Vancomycinextracted from a complex sample matrix with the activated Gibbs reagentin accordance with another embodiment of the present invention;

FIG. 10 depicts absorption characteristics of the reaction of freeVancomycin (A) and protein-bound Vancomycin (B) extracted from a numberof complex sample matrices comprising different protein concentrationswith the activated Gibbs reagent in accordance with another embodimentof the present invention;

FIG. 11 is a plot of the ratios of free and protein-bound Vancomycinconcentrations as extracted from the complex sample matrices shown inFIG. 10;

FIG. 12 depicts an absorption characteristics comparison of the Gibbsreaction product of Vancomycin and other phenolic compounds extractedfrom whole serum samples;

FIG. 13 depicts an absorbance spectrum of the Gibbs reaction product ofVancomycin and Propofol; and

FIG. 14 is a flow chart of another method of the present invention.

DETAILED DESCRIPTION OF THE DRAWINGS

It should be understood that the Figures are merely schematic and arenot drawn to scale. It should also be understood that the same referencenumerals are used throughout the Figures to indicate the same or similarparts.

The invention can be applied to situations where a glycopeptideantibiotic analyte of interest such as Vancomycin has to be extractedfrom a complex, in particular a biological, sample. It is in particularapplicable to the extraction of glycopeptide antibiotic species fromblood or food samples. In these samples, some of the analyte or analytesof interest contained in the sample will typically be bound toconstituents of the sample, for example, blood cells or proteins. Inorder to measure the concentration of the analyte or analytes in thesample, the analyte or analytes will often have to be extracted fromthese constituents during a sample preparation step prior to the actualdetection or measurement step. While the invention will be illustratedprimarily with respect to extraction of a glycopeptide antibiotic suchas Vancomycin from blood samples, it is equally applicable to othercomplex sample matrices, e.g. samples containing matter such as cells orsimilar structures.

In general, embodiments of the present invention employ a combination ofdilution, osmotic lysis and mechanical lysis to break up cells and makethe analyte of interest bound to the cell components available foranalysis. In order to achieve this aim, the sample is initially dilutedusing a lysing agent, for example de-ionised water, solutions containingdetergents, urea, acids or combinations thereof; the actual choice oflysing agent will depend on the characteristics of the sample andsuitable agents are known to those trained in the art of cell lysis. Thedilution step will result in the cells contained in the blood sampletaking on components of the lysing agent (e.g. water) and thereforeswelling or breaking up. The cells contained in the sample willtherefore either burst and release their content, or will be in a morefragile state (e.g. in the form of erythrocyte ghosts in the example ofosmotic lysis by the dilution into water).

Following a delay stage (typically of the order of seconds to severalminutes), the diluted sample will then be passed through a solid phaseextraction cartridge. An additional pre-filter may also be incorporatedjust before the SPE cartridge. The advantage of such a pre-filter overthe prior art is that it may also be used for mechanical lysis andexposed to subsequent extraction of the analyte of interest using asuitable eluent as it is placed in the fluid lines of the apparatus ofthe present invention.

The shear forces introduced by the action of the SPE cartridge or thefilter/SPE cartridge combination serves to increase the lysis efficiencyby breaking up the intact erythrocytes, erythrocyte ghosts and othercell fragments that remained after the dilution into the lysis agent.The composition of the sorbent in the cartridge or pre-filter is chosensuch that it is able to bind the analyte of interest, i.e. aglycopeptide antibiotic such as Vancomycin, in preference to thecomponents of the sample matrix. The combined action of diluting thesample with a lysing agent and lysing the remaining cells by shear forceincreases the availability of the analyte of interest for extractiononto the sorbent of the solid phase extraction column.

There is also the possibility of some components of the sample matrix(e.g. unlysed cells or fragments thereof) being accumulated on top ofthe sorbent bed or pre-filter and not being passed through the sorbent.In a subsequent step, the sorbent cartridge is washed using one or moresuitable washing agent(s). The choice of washing agent will depend onthe analyte of interest; the washing agent should preferentially removeinterferents from the sorbent and sample material accumulated on top ofthe sorbent bed, while leaving the analyte of interest bound to thesorbent or filter material.

Additionally, in one embodiment of the invention, the washing agent willassist in the break up of the accumulated cell debris on the sorbent bedand increase the availability of the analyte for extraction.

Following the washing step, the analyte of interest is eluted from thesorbent, pre-filter and/or the materials accumulated on top of thesorbent/filter material. This elution step employs a suitable extractionagent that is able to dissolve the analyte of interest from the sorbentor filter material as well as from the material left behind on top ofthe sorbent or filter. As will be demonstrated in more detail later, itmay be possible to selectively extract a particular fraction of theanalyte of interest from the sorbent using appropriate eluents; forinstance, an analyte of interest bound only to sorbent material may beremoved using a different eluent compared to an analyte of interestbound to sorbent material and/or cellular material present in thecomplex sample matrix.

The eluent is then passed into a detection chamber where the analyte ofinterest is detected or the amount of the analyte of interest in theeluent is quantified. A range of detection or measurement techniques areavailable depending on the analyte of interest. For example, in the caseof Vancomycin as an example of a glycopeptide antibiotic, colorimetricor electrochemical detection techniques may be used.

The glycopeptide antibiotic of interest is detected as a reactionproduct of its reaction the activated Gibbs reagent. The proposedreaction scheme 1 is depicted below for glycopeptide antibioticscontaining phenol moieties such as Vancomycin. It is noted that this isa proposed reaction scheme only that has not been verified such that analternative reaction scheme may apply.

This however does not influence the teachings of the present inventionas it has been demonstrated (see examples) that the Gibbs reaction canbe effectively performed between Vancomycin and the activated Gibbsreagent when performing the Gibbs reaction under alkaline conditions(i.e. pH>7) using an excess of the Gibbs reagent wherein the excesspreferably is limited to five times the molar amount of the Gibbsreagent relative to the molar amount of the glycopeptide antibiotic toavoid the formation of such different products and fragments.

One non-limiting embodiment of the present invention is described inmore detail in FIG. 1 in conjunction with FIG. 2, which shows anapparatus 20 capable of implementing this method. This embodimentcomprises the following steps, some of which are optional as indicatedby the dashed boxes in FIG. 1. It should be noted that where referenceis made in the following to water as an eluent or washing agent, this isintended to refer to water per se as well as to aqueous buffers.

1. Introducing a Sample Volume to the Fluidic System

The sample introduced into the apparatus 20 in any suitable manner, e.g.either in a manual or automated fashion.

If concentration measurements are being made by the apparatus 20, it ispreferred that a known volume of this sample is made available forfurther processing by the apparatus 20. Metering a known volume from thesample may be achieved using a sample metering module 21. An examplesuitable implementation of a method of delivering a known volumeincludes, but is not limited to, a 6 port, 2 position rotary valveconnected to the fluidic system. This valve can be used to switch alength of tubing 22 containing a known volume of the sample of interestinto the rest of the fluidic system. This is shown in FIG. 2 as valveposition A. Other methods of introducing a known sample volume to afluidic system will be known to those who are skilled in the art.

Alternatively, a known volume of sample may be introduced eithermanually or automatically in the first place.

In one particular embodiment, the sample is a sample of body fluids, forexample, blood, plasma, serum, urine, saliva, cerebral fluids and so on.In another embodiment, the sample may be a food product or food productextract, e.g. milk or a meat extract.

In a preferred embodiment of the invention, the sample contains the drugVancomycin as the analyte of interest, as the inaccurate determinationof this drug in such samples can seriously impair the effectiveness ofthe drug administration regime, and can cause micro-organisms becomingresistant to such antibiotics as explained in more detail above. Themethod and the device of the present invention enable the extraction ofglycopeptide antibiotics with phenol functional groups, e.g. Vancomycin,from such samples with unparalleled accuracy, which facilitates thedetection of such glycopeptide antibiotics e.g. through colorimetry byperforming the Gibbs reaction on the glycopeptide antibiotics as will beexplained in more detail later.

2. Dilution of the Sample into an Lysing Agent

The blood sample or parts thereof, for example, the metered volume ofthe sample, are subsequently mixed with a lysing agent, which may beintroduced into the apparatus 20 via tubing 23. The lysing agent ischosen dependent on the sample and the subsequent extraction step. Itcan, for example, be de-ionised water, aqueous salt solutions with anosmolarity lower than that of the sample, solutions containing ammoniumchloride, detergents, urea, acids or combinations thereof. In situationswhere the analyte of interest (Vancomycin) is subsequently to beextracted by reverse phase, osmotic lysis by dilution into de-ionisedwater is preferred.

The dilution itself may be achieved through the use of a known length oftubing that bypasses the sample metering module, such as the tubing 24shown in FIG. 2. The relative resistances of the sample metering andbypass lines are chosen to allow for dilution of the sample, for examplea dilution between a 1-fold and 100-fold dilution of the sample(preferably 15-fold). The exact dilution of a given sample will varydepending on its viscosity. Methods of tuning the relative resistanceswill be known to those who are skilled in the art, and may include, butare not limited to, controlling the length and internal diameter of thetubing and the incorporation of a flow restrictor. Such principles areknown per se e.g. from the field of microfluidics and will not beexplained in further detail for the sake of brevity only.

3. Providing Time for the Sample and the Lysis Agent to Mix and Reactwith each Other

In order to enable the lysis agent and the sample to interact with eachother, the diluted sample is given some time before it is passed to themechanical lysis stage 27 in FIG. 2. This time period depends on thesample being investigated and the nature of the lysis method beingemployed, but is preferentially of the order of one second to severalminutes. This period should be sufficiently long for any cells containedin the diluted sample to be fragile enough for the mechanical lysisstage.

This delay period may be generated by a delay stage, such as delay stage25 as shown in FIG. 2. This delay stage 25 preferably consists of adefined length of tubing, which, in conjunction with a controlled rateof fluid flow, allows the sample to incubate for a period of time.

4. Mechanical Lysis of the Diluted Sample

After the delay stage 25, the diluted sample is passed across amechanical lysis stage 27. The purpose of this mechanical lysis stage 27is to mechanically lyse (break up) the remainder of the cells, theerythrocyte ghosts and the cell membrane fragments by shear forces. Theprimary purpose of this step is to facilitate the release of theantibiotic of interest, e.g. Vancomycin, from the cells, cell componentsand cell membranes, thereby making it more available for subsequentextraction onto the sorbent. The secondary purpose of this stage is toensure that large intact cells and cell fragments that remain after theinitial chemical/osmotic and mechanical lysis steps are retained untilthey can be broken up sufficiently for efficient analyte extraction fromthem during subsequent steps (e.g. steps 6-8).

In one preferred embodiment, the mechanical lysis stage 27 consists of asolid-phase extraction cartridge containing a suitable sorbent. Thesorbent may be in powdered or in co-sintered form. It may be held inplace using a suitable frit. Preferably, the solid phase extractionsorbent should also be capable of extracting the analyte of interestfrom the lysate. In another embodiment, a filter 26 or a combination ofa filter 26 and a solid phase extraction sorbent may be used, with thefilter 26 providing a pre-filtering step 13 to aid the mechanical lysisby also retaining intact cells and large cell fragments. Porousmaterials, including, but not limited to, glass/metallic/polymer fibres,powders or sintered polymer membranes may be used as filters for themechanical lysis. Other methods of producing porous materials will beknown to those who are skilled in the art. Passing the diluted samplethrough the sorbent and/or porous material will apply shear forces tothe cells and cell fragments which will break up the cells. Othermethods of applying shear force will be known to those who are skilledin the art.

The diluted and lysed sample is then brought into contact with asuitable sorbent or filter material. The composition of the sorbent orfilter material is chosen such that it is able to bind the glycopeptideantibiotic of interest, e.g. Vancomycin, in preference to the dilutedand lysed sample. The purpose of this step is to extract the analyte ofinterest from the sample and bind it to the sorbent or filter material.

In one embodiment of the invention, the sorbent or filter 26 and themechanical lysis stage 27 are combined in one unit. In a preferredembodiment of the invention, the sorbent or filter is used as the porousmaterial in the mechanical lysis stage.

For example, in the case of Vancomycin being the analyte of interest, areverse-phase sorbent cartridge is preferred, made from materials knownto those trained in solid phase extraction and including materials, suchas C18, C8 and Strata-X.

In a preferred embodiment of the invention, the sorbent is contained ina solid-phase extraction cartridge.

5. Purging of the Sample Metering and Bypass Sections

After all the diluted sample has been transferred to the mechanicallysis/analyte extraction module via the delay stage, the samplemetering, bypass, delay and mechanical lysis sections can then beflushed with a large excess of the lysing agent to ensure that allsedimented cells are purged from these lines and transferred to theextraction stage. To this end, the valve 21 is placed in position B asshown in FIG. 2.

6. Washing Step

The sorbent cartridge and/or filter may then be washed using one or moresuitable washing agent(s). The purpose of this washing step is to removeany substance which may interfere with the subsequent detection of theanalyte of interest from the material adsorbed in the sorbent cartridgeor filter. A first washing step with water may be applied to removeweakly bound impurities. This washing step may be repeated if necessary,e.g. two washing steps with 1 ml de-ionized water may be applied forthis purpose. The washing step is found to significantly improve theaccuracy of the determination of the free and bound fraction of theglycopeptide antibiotic (Vancomycin) by suppressing unwanted Gibbsreactions, e.g. with weakly bound impurities, in particular proteins.

This may be followed by a second washing step with pure organicsolvents, e.g. methanol, to remove lipophilic and hydrophobic speciesthat may interfere with the Gibbs coupling reaction and may lead tocross-contamination, such as, but not limited to, propofol.

The second washing step may be collected in case the concentration ofthe free (unbound) glycopeptide antibiotic such as Vancomycin is to bedetermined using an organic solvent such as methanol as eluent aspreviously explained. In this case, although it may be difficult tocompletely rule out unwanted (competing) Gibbs reactions in thisscenario (see example 5 below), the use of excess amount of Gibbsreagent ensures that the concentration of the unbound glycopeptideantibiotic can be accurately determined by ensuring that a sufficientamount of Gibbs reagent remains to react with the glycopeptideantibiotic.

The first and second washing steps may be repeated if necessary, e.g. byperforming a number of first washing steps prior to performing a numberof second washing steps. Only the first of the second washing steps mayrequire collecting to determine the concentration of the free (unbound)glycopeptide antibiotic such as Vancomycin although the eluents ofmultiple second washing steps may be combined for the same purpose.

7. Extraction of the Analyte of Interest from the Sorbent or Filter

Standard techniques are then used to extract the analyte of interestfrom the sorbent or filter and the sample materials left behind on topof the sorbent and/or filter. For example, a known volume of a suitablesolvent which preferentially dissolves/binds the analyte of interestwith respect to the sorbent and/or filter may be passed through thesorbent and/or filter to remove the analyte of interest from the sorbentand/or filter. These techniques will be known to those who are skilledin the art.

In the case of Vancomycin being the analyte of interest, methanol may beused to extract unbound Vancomycin from a sorbent such as a reversephase sorbent material, e.g. Strata-X, whereas a volume of 1 part (byvolume) water and two parts (by volume) methanol may be used to extractVancomycin bound to serum constituents of the complex sample matrix froma sorbent such as a reverse phase sorbent material, e.g. Strata-X; othersuitable extraction agents and methods of extraction are known to thosetrained in the art of solid phase extraction.

In the special case of separate quantification of free and bound, e.g.protein-bound Vancomycin concentrations, a volume of organic solventsuch as methanol may be used as a first eluent and collected for thefree concentration quantification. This may then be followed by the useand collection of a volume of 1 part (by volume) water and two parts (byvolume) methanol, which removes the bound fraction of the Vancomycin ofthe sorbent and/or filter.

8. Detection or Concentration Measurement of the Analyte of Interest inthe Eluent

The eluent from the sorbent and/or filter is passed into a detectionstage 28 where the concentration of the analyte in the extract, andhence the original concentration in the blood sample, can then bedetermined using a suitable detection system. The detection systemrequired will vary depending on the analyte of interest. In aparticularly preferred embodiment, the detection stage 28 comprises acolorimeter for determining the colorimetric spectrum of the Gibbsreaction product with the phenol moiety-containing antibiotic ofinterest. The colorimetric detection of the Gibbs reaction productinvolving propofol is disclosed by McGaughran et al in Journal ofClinical Monitoring and Computing, vol. 20, no. 5, pp. 381-381, 2006,which teachings apply mutatis mutandis. Other suitable methods foranalyte detection may include, but are not limited to, electrochemical,fluorescent or gravimetric approaches. Other methods of detection willbe known to those who are skilled in the art. It has been found (seebelow) that the reaction between the glycopeptide antibiotic and theGibbs reagent is particularly successful if an excess Gibbs reagent isused. About a 5-fold excess is particularly preferred as previouslyexplained.

The generated spectrum may be collected by a processor 29, whichdetermines the glycopeptide antibiotic concentration in the complexsample matrix from the intensity of the absorption in the range of580-600 nm and preferably at 589 nm for Vancomycin or anotherappropriate wavelength in case of the Gibbs reaction product of theactivated Gibbs reagent with another phenol functional group containingglycopeptide antibiotic. The processor 29 may form part of the detectionstage 28 or may be separate to the detection stage 28. Consequently, afully automated method and apparatus for determining the concentrationof a phenol moiety-containing glycopeptide antibiotic such as Vancomycinin a complex matrix within minutes at low cost is provided.

It is noted that it will be clear that the apparatus of the presentinvention may comprise other useful features, such as one or morereservoirs in fluid communication with the detection stage 28 forcontaining the respective reagents for performing the Gibbs reaction onthe eluent. Respective metering devices may be present between thereservoirs and the detection stage 28 for metering the amounts ofreagents fed into the detection stage 28. Such metering devices may forinstance be controlled by the processor 29 or another processor.

The proof of concept of the successful extraction of free (unbound) andbound Vancomycin from a whole serum sample using the method of thepresent invention will be demonstrated with the aid of the followingexamples. It should be understood that these examples are not intendedto limit the scope of the invention and that variations to this example,e.g. the use of another sample type or another glycopeptide antibioticare feasible without departing from the teachings of the presentinvention.

Synthesis Example 1

In order to demonstrate that the present inventors have developed aprotocol under which glycopeptide antibiotics such as Vancomycin canreact with the activated Gibbs reagent, 0.8 mM of Vancomycin wasdissolved in a borate buffer (0.4 M NaOH) and reacted with an equimolaramount of the activated Gibbs reagent in a mixture of methanol andborate buffer under alkaline conditions. FIG. 3 shows the UV-vis spectraof the two starting products, Vancomycin (i) and the activated Gibbsreagent (ii), and the novel purple/magenta coupling product (iii). Themaximal absorbance wavelength (λ_(max)) of the newly formed couplingproduct is 589 nm (highlighted by the box in FIG. 3) and itsconcentration can be calculated via the Beer-Lambert law. The reactionwas repeated with increasing amounts of Gibbs reagent(Vancomycin:activated Gibbs reagent molar ratios 1:2, 1:3, 1:4, 1:5 and1:65) to demonstrate that a completed coupling reaction is achieved whenusing excess Gibbs reagent.

The top panel of FIG. 4 shows the theoretical prediction of the isotopicpattern of mass from the novel coupling product (C₇₂H₇₆Cl₄N₁₀O₂₅) basedon 1:1 stoichiometric ratio of Vancomycin:Gibbs under alkalineconditions. The bottom pane of FIG. 4 shows the experimentally measuredmass spectrum of the novel reaction product. The mass spectrum wasdetermined using an Agilent 1100 series G1946D with an electrosprayionisation (ESI) probe from Agilent (Agilent, Santa Clara, Calif.,U.S.A.) after purification of the reaction product using an integratedHPLC instrument (Agilent 1100 Series HPLC System). As can be seen, thetheoretical and experimental mass spectra are in excellent agreement,thus clearly suggesting the formation of the Vancomycin:Gibbs reactionproduct.

FIG. 5 shows the several ¹H-NMR spectra including starting materialsVancomycin and Gibbs reagent both under alkaline conditions and severalreactions with the increasing equivalents of Gibbs reagent used in thesynthesis example 1. The NMR spectra were recorded on an Avance III 600Cryo NMR spectrometer from Bruker (Bruker, Billerica, Mass., U.S.A.) in⅓ D₂O+⅔ MeOD by volume with 0.4 M NaOD in D₂O. The water peak was usedfor PPM calibration. The disappearance of the two peaks just below 6 ppmhighlighted with a dotted box with increasing Gibbs equivalent providesclear evidence that Gibbs is coupling to the aromatic ring indicated inReaction Scheme 1. The assignment of all other Vancomycin protons whichremained unchanged due to the Gibbs addition is in good agreement withAntipas et al., Journal of Pharmaceutical Sciences 88(6), pages 742-750,2000 and Pearce and Williams, J. Chem. Soc., Perkin Trans. 2(1), pages153-157, 1995, which disclose the 1H NMR spectrum of Vancomycin.

Example 1

A 1 ml whole serum sample spiked with Vancomycin is applied to a 30 mg/1ml reversed phase Strata-X™ SPE column from Phenomenex®. The column iswashed firstly with 2 ml water to remove weakly bound impurities;followed by a wash with approximately 2 ml of pure organic solvents suchas, but not limited to, methanol, to remove lipophilic respectivelyhydrophobic species, which may interfere with the coupling reaction andmay lead to cross-contamination, such as, but not limited to, propofol.The Vancomycin bound to serum constituents present in the whole serumsample is then eluted from the SPE column using a known volume (0.5 ml)of a mixture of one-third water and two-thirds methanol (both byvolume).

A 350 μl fraction of the elute is then transferred into a vial. Then 50μl of 3.625 mM Gibbs in methanol is added, followed by 40 μl of 0.4 Msodium hydroxide in borate buffer or water, which initiates the couplingreaction and results in an immediate colour change. After a shake,typically 350 μl of the mixture is transferred into a cuvette and itsspectra is measured via a spectrophotometer.

Comparative Example 1

Comparative example 1 is the same as example 1 with the difference thata 1 ml whole serum sample without Vancomycin is applied to a 30 mg/1 mlreversed phase Strata-X™ SPE column.

FIG. 6 shows the typical UV/vis spectra of Gibbs labelling of two SPEelutes from serum, sample (a) spiked with Vancomycin from example 1, anda reference (b) without Vancomycin from comparative example 1. Thespectrum (c) is the difference between the sample and referencespectrum. The absorbance at 589 nm suggests that Vancomycin can berecovered out of the complex sample matrix via Strata-X and subsequentlylabelled with Gibbs reagent in a comparable manner and in very goodagreement with synthesis example 1 and FIG. 3. The correspondingVancomycin concentration can then be calculated via the Beer-Lambertlaw.

Example 2

In order to verify the accuracy of the present method, whole bloodserums were spiked with different Vancomycin concentrations (1.2 μM, 2.4μM, 4.8 μM, 9.5 μM, 14.3 μM and 21.5 μM respectively) and passed overthe assay as described in example 1. Two fractions of each elute wereindependently labelled with the Gibbs reagent to get an estimation ofthe accuracy of the novel Vancomycin assay. FIG. 7 shows the UV-visdifferential absorption spectra of the various eluents after completingthe Gibbs reaction against the reference of comparative example 1, whichclearly demonstrates that with this novel assay detection of Vancomycinin the therapeutic window, 3-29 μM, is achievable.

This is further corroborated by FIG. 8, which depicts a linear fit ofthe eluent concentrations as obtained from the differential absorptionspectra at 589 nm using a fitting function y=a+b*x, in which a was foundto be −0.042±0.004; b was found to be 0.01±0.004 with a R² value of0.994, thus demonstrating an excellent fit between the spike levels andthe concentrations obtained from the eluent extracted from the serumsamples including these spike levels.

Example 3

A 1 ml whole serum sample (VVHS) spiked with Vancomycin is applied to a30 mg/1 ml reversed phase Strata-X™ SPE column from Phenomenex®. Thecolumn is washed firstly with 2 ml water to remove weakly boundimpurities. This washing step may be repeated if necessary. Then a knownvolume (1 ml) of an organic solvent such as, but not limited to,methanol is used to elute the free Vancomycin fraction from the sorbent.This optionally may be followed by another organic washing step, e.g.another washing step with an organic solvent, which may be the samesolvent or a different solvent as used in the elution step. The boundVancomycin fraction is then eluted from the SPE column using a knownvolume (0.5 ml) of a mixture of one-third water and two-thirds methanol(by volume).

A 350 μl fraction of each elute is then transferred into separate vials.Then 50 μl of 3.625 mM Gibbs in methanol is added to each vial, followedby 40 μl of 0.4 M sodium hydroxide in borate buffer or water, whichinitiates the coupling reaction and results in an immediate colourchange. After a shake, typically 350 μl of each mixture is transferredinto a cuvette and their spectra are measured via a spectrophotometer.

FIG. 9 shows the differential UV-vis spectra of the free and boundfractions of the same WHS sample spiked with 29 μM Vancomycin, whichwere subtracted from comparative example 3. The maximal absorbancewavelength (λ_(max)) of the newly formed coupling product is 589 nm(highlighted by a vertical line in the graph) and the corresponding freeand bound concentrations can be calculated via the Beer-Lambert law.

Comparative Example 2

Comparative Example 2 is the same as example 3 with the difference thata 1 ml whole serum sample without Vancomycin is applied to a 30 mg/1 mlreversed phase Strata-X™ SPE column.

Example 4

In order to verify the accuracy of the method presented in Example 3,samples with several different serum protein concentrations, hereinhuman serum albumin (HSA) (600 μM, 300 μM, 150 μM, 75 μM and 0 μM)dissolved in water, as well as whole human serum (WHS) were spiked withthe same Vancomycin concentration (29 μM). It is noted that 600 μMcorresponds to the concentration of serum albumin in whole human serum.These samples were passed over the assay as described in Example 3.Collection from both elutes, the organic elute and the mixture ofone-third water and two-thirds methanol (by volume) elute, wereindependently labelled with the Gibbs reagent to get an estimation ofthe accuracy of the novel free and bound Vancomycin assay.

FIGS. 10A and B show the differential absorbencies of the organic (A)and the mixed water/organic (B) elutes respectively for the severalprotein concentrations and whole human serum (WHS) captured via UV-visspectroscopy. The corresponding absorbencies values for free and boundat the maximal absorbance wavelength (λ_(max)=589 nm) of the novelcoupling product were used to calculate the percentages of Vancomycin[%] comprised in the free and bound fractions, which is graphicallyillustrated in FIG. 11.

For commercially available whole human serum typically 63±12% was foundto be free and 37±2% to be bound. The errors are derived from thestandard deviation from three independent measurements (n=3) indicatedby the error bars in the FIG. 11.

Example 5

In order to demonstrate the specificity of the extraction protocol forglycopeptide antibiotics with one or more phenolic moieties such asVancomycin, four whole serum samples were prepared with 0.1 μM Dopamine,132 μM Paracetamol and 56.1 μM Propofol as potentially interferingphenolic compounds and 29 μM Vancomycin. The column is firstly twicewashed with 2 ml water to remove weakly bound impurities. Then a knownvolume (1 ml) of methanol is used to elute the free Vancomycin fractionfrom the sorbent. This optionally may be followed by another organicwashing step, e.g. another washing step with methanol. The boundVancomycin fraction is then eluted from the SPE column using a knownvolume (0.5 ml) of a mixture of one-third water and two-thirds methanol(by volume).

After each washing/elution step, the washing agent/eluent was collectedand reacted with the Gibbs reagent using the reaction conditions ofExample 1 and the reaction product was evaluated as explained inExample 1. The results are shown in FIG. 12.

It can be seen that the first and second washing steps with water removenegligible amounts of the phenolic compounds from the sorbent material,but that the first methanol eluent removes the unbound fraction ofVancomycin as previously demonstrated as well as at least some of thePropofol. The optional subsequent washing step with methanol furtherremoves some of the Vancomycin, such that the first eluent may becombined with the methanol used in this washing step to further improvethe accuracy of the unbound Vancomycin determination. The second eluent(methanol/water) removes the bound fraction of Vancomycin at excellentspecificity.

It therefore has been demonstrated that glycopeptide antibiotics such asVancomycin can be removed with good selectivity from the sorbentmaterial using the proposed extraction protocol in the presence of otherphenolic compounds. It is noted that Propofol shows some interference inthe first eluent (methanol) but this does not affect the determinationof the bound fraction of the glycopeptide antibiotic, which is derivedfrom the second eluent.

In order to avoid potential interference of Propofol in thedetermination of the unbound fraction of the glycopeptide antibiotic,the following measures may be considered. Since Propofol does notnaturally occur in patients, the unbound fraction of a glycopeptideantibiotic such as Vancomycin may be determined when the patient is notunder the influence of Propofol. Alternatively, the Propofolconcentration may be determined independently on a separate extractionassay, e.g. using the protocols disclosed in WO 2012/049486 A1 such thatthe contribution of Propofol to the overall determination can besubtracted to yield the unbound glycopeptide antibiotic concentration.

Moreover, since the Propofol/Gibbs reaction product is blue andconsequently has a λ_(max) of 595 nm and Vancomycin/Gibbs reactionproduct is purple/magenta with λ_(max) of 589 nm, see FIG. 13, measuringthe UV/vis spectrum over an appropriate spectral range instead of at afixed wavelength (i.e. at 589 nm) may allow for the extraction of thepropofol contribution from the overall determined concentration. Forinstance, by measuring the absorbance in a window from for instance from500-700 nm, the shoulder appearing above 600 nm that is caused by theconcentration of Propofol:Gibbs reaction product present in the samplecan be used to calculate, e.g. subtract, the contribution of thePropofol:Gibbs reaction product to the absorbance at 589 nm.

As can be seen from FIG. 6-13, it is possible to accurately determinethe Vancomycin levels within its therapeutic window, which for instancehas been identified in FIG. 8 by the shaded area. This can beadvantageously used to personalized administration regime of anantibiotic containing a phenol moiety such as Vancomycin for a patient.An example of such a method of determining a personalized administrationregime of an antibiotic containing a phenol moiety such as Vancomycinfor a patient using the present invention is shown in FIG. 14.

In step 72, the concentration of the antibiotic in a complex samplematrix of said patient is determined in accordance with an embodiment ofthe method of the present invention as described in detail above withthe aid of FIG. 1. In step 74, the determined concentration is comparedagainst the administered amount of the phenol moiety-containingantibiotic, e.g. Vancomycin, to derive the metabolic characteristics ofthe antibiotic in the patient. In particular, it may be determined ifthe determined concentration still falls within the therapeutic windowof the antibiotic.

In an embodiment, step 72 may comprise periodically determining theconcentration of the antibiotic in a complex sample matrix of saidpatient before deriving the metabolic characteristics of the antibioticin the patient, as such periodic measurements can be used to determine atrend in the determined concentrations, thus giving further insight themetabolic characteristics of the antibiotic in the patient. Forinstance, such a trend may indicate the metabolic rate for anadministered amount of the antibiotic.

In step 76, the determined metabolic characteristics of the antibioticin the patient are used to design a personalized antibioticadministration regime for the patient to ensure that the amount of theantibiotic in the patient's metabolism remains within the therapeuticwindow, thus significantly reducing the risk that the treatment isineffective, with the potential grave consequence of the microbialtarget of the treatment becoming resistant to the administeredantibiotic, e.g. Vancomycin.

At this point it is noted that the Gibbs reaction involving the phenolfunctional group containing antibiotic, e.g. Vancomycin preferably isperformed following the extraction of the antibiotic from the sorbentand/or filter material, i.e. in the eluent.

It should be noted that the above-mentioned embodiments illustraterather than limit the invention, and that those skilled in the art willbe able to design many alternative embodiments without departing fromthe scope of the appended claims. In the claims, any reference signsplaced between parentheses shall not be construed as limiting the claim.The word “comprising” does not exclude the presence of elements or stepsother than those listed in a claim. The word “a” or “an” preceding anelement does not exclude the presence of a plurality of such elements.The invention can be implemented by means of hardware comprising severaldistinct elements. In the device claim enumerating several means,several of these means can be embodied by one and the same item ofhardware. The mere fact that certain measures are recited in mutuallydifferent dependent claims does not indicate that a combination of thesemeasures cannot be used to advantage.

What is claimed is:
 1. A method of determining a concentration of aglycopeptide antibiotic containing a phenol moiety in a complex samplematrix, the method comprising: extracting the glycopeptide antibioticfrom a metered portion of the complex sample matrix by: exposing saidmetered portion of the complex sample matrix to an extraction materialhaving an affinity with the glycopeptide antibiotic; and exposing theextraction material to a metered portion of a first eluent being a firstorganic solvent or a second eluent being a second organic solvent/watermixture for releasing the glycopeptide antibiotic from the extractionmaterial; and determining a concentration of the glycopeptide antibioticby: adding an excess amount of Gibbs reagent (2,6dichloroquinone-4-chloroimide) relative to the amount of theglycopeptide antibiotic containing a phenol moiety to the meteredportion of the first or second eluent; activating the Gibbs reagentunder alkaline conditions and, after the reaction between the activatedGibbs reagent and the glycopeptide antibiotic has stabilized; detectingthe reaction product of the activated Gibbs reagent and the glycopeptideantibiotic in said metered portion of the first or second eluent; anddetermining a concentration of the glycopeptide antibiotic in thecomplex sample matrix from the detected reaction product, saidconcentration being: the concentration of unbound glycopeptideantibiotic in the complex sample matrix in case of the first eluent; orthe concentration of bound glycopeptide antibiotic in the complex samplematrix in case of the second eluent, and wherein the step of exposingthe extraction material to a metered portion of a first or second eluentis performed with the first eluent and the concentration of theglycopeptide antibiotic in the complex sample matrix from the detectedreaction product is the concentration of unbound glycopeptide antibioticin the complex sample matrix, the method further comprising the step ofexposing the extraction material to a metered portion of the secondeluent and the concentration of the glycopeptide antibiotic in thecomplex sample matrix from the detected reaction product is theconcentration of bound glycopeptide antibiotic in the complex samplematrix.
 2. The method of claim 1, wherein the first and/or the secondorganic solvent is methanol.
 3. A method of determining a concentrationof a glycopeptide antibiotic containing a phenol moiety in a complexsample matrix, the method comprising: extracting the glycopeptideantibiotic from a metered portion of the complex sample matrix by:exposing said metered portion of the complex sample matrix to anextraction material having an affinity with the glycopeptide antibiotic;and exposing the extraction material to a metered portion of a firsteluent being a first organic solvent or a second eluent being a secondorganic solvent/water mixture for releasing the glycopeptide antibioticfrom the extraction material; and determining a concentration of theglycopeptide antibiotic by: adding an excess amount of Gibbs reagent(2,6 dichloroquinone-4-chloroimide) relative to the amount of theglycopeptide antibiotic containing a phenol moiety to the meteredportion of the first or second eluent; activating the Gibbs reagentunder alkaline conditions and, after the reaction between the activatedGibbs reagent and the glycopeptide antibiotic has stabilized; detectingthe reaction product of the activated Gibbs reagent and the glycopeptideantibiotic in said metered portion of the first or second eluent; anddetermining a concentration of the glycopeptide antibiotic in thecomplex sample matrix from the detected reaction product, saidconcentration being: the concentration of unbound glycopeptideantibiotic in the complex sample matrix in case of the first eluent; orthe concentration of bound glycopeptide antibiotic in the complex samplematrix in case of the second eluent, and wherein the second eluent is amethanol/water mixture in a 2:1 ratio by volume.
 4. A method ofdetermining a concentration of a glycopeptide antibiotic containing aphenol moiety in a complex sample matrix, the method comprising:extracting the glycopeptide antibiotic from a metered portion of thecomplex sample matrix by: exposing said metered portion of the complexsample matrix to an extraction material having an affinity with theglycopeptide antibiotic; and exposing the extraction material to ametered portion of a first eluent being a first organic solvent or asecond eluent being a second organic solvent/water mixture for releasingthe glycopeptide antibiotic from the extraction material; anddetermining a concentration of the glycopeptide antibiotic by: adding anexcess amount of Gibbs reagent (2,6 dichloroquinone-4-chloroimide)relative to the amount of the glycopeptide antibiotic containing aphenol moiety to the metered portion of the first or second eluent;activating the Gibbs reagent under alkaline conditions and, after thereaction between the activated Gibbs reagent and the glycopeptideantibiotic has stabilized; detecting the reaction product of theactivated Gibbs reagent and the glycopeptide antibiotic in said meteredportion of the first or second eluent; and determining a concentrationof the glycopeptide antibiotic in the complex sample matrix from thedetected reaction product, said concentration being: the concentrationof unbound glycopeptide antibiotic in the complex sample matrix in caseof the first eluent; or the concentration of bound glycopeptideantibiotic in the complex sample matrix in case of the second eluent,and further comprising exposing the extraction material to at least onewashing step prior to exposing the extraction material to said meteredportion of the first or second eluent to remove weakly bound impuritiesfrom the extraction material.
 5. The method of claim 1, wherein thecomplex sample matrix comprises cellular material, and wherein the stepof exposing said metered portion of the complex sample matrix to anextraction material having an affinity with the glycopeptide antibioticcomprises: providing a known quantity of the complex sample matrix;diluting said known quantity with a lysing agent, said lysing agenthaving an osmotic pressure lower than the osmotic pressure of thecellular material, to provide destabilized cellular material; andsubsequently feeding the diluted known quantity through a filteringstage comprising the extraction material after a predetermined amount oftime to separate the glycopeptide antibiotic from the destabilizedcellular material by selectively adhering the glycopeptide antibiotic tothe extraction material.
 6. The method of claim 5, wherein theextraction material comprises a sorbent material for mechanically lysingthe cellular material, said sorbent material having an affinity forbinding the glycopeptide antibiotic.
 7. The method of claim 6, whereinsaid sorbent material is a reverse phase sorbent material.
 8. The methodof claim 1, wherein said detecting step comprises electrochemically orcolorimetrically detecting the reaction product of the activated Gibbsreagent and the glycopeptide antibiotic.
 9. The method of claim 8,wherein said colorimetrically detecting comprises measuring theintensity of an absorption in a range of 580-600 nm.
 10. The method ofclaim 1, wherein the glycopeptide antibiotic is Vancomycin.
 11. Themethod of claim 1, wherein the complex sample matrix is one of blood,plasma and serum or is obtained from a food product.
 12. A method ofproviding a personalized administration regime of a glycopeptidesantibiotic containing a phenol moiety for a patient, the methodcomprising: determining the concentration of the glycopeptide antibioticin a complex sample matrix of said patient in accordance with the methodof any one of claims 1, 3 or 4; deriving metabolic characteristics ofthe glycopeptide antibiotic for said patient from the determinedconcentration of the glycopeptide antibiotic in said complex samplematrix; and providing said personalized glycopeptide antibioticadministration regime based on said metabolic characteristics.
 13. Themethod of claim 12, wherein said determining step comprises periodicallydetermining the concentration of the glycopeptide antibiotic, andwherein said deriving step comprises deriving said metaboliccharacteristics from a trend in the periodically determinedconcentrations.
 14. The method of claim 7, wherein the reverse phasesorbent material is C8, C12, C18 or Strata-X.
 15. The method of claim 9,wherein the colorimetrically detecting comprises measuring the intensityof an absorption at 589 nm.
 16. The method of claim 11, wherein the foodproduct is milk or meat.